Human Breast Milk and breast feeding represent the uncontested gold standard in terms of infant nutrition. Infant formulae that serve as a substitute for or complement to human breast milk should satisfy the nutritional requirements of infants, have an acceptable taste and be hypoallergenic and tolerogenic (i.e. able to induce oral tolerance) when targeted to infants at risk of allergy. Induction of oral tolerance to cow's milk has been described in EP0827697. It is known that allergies to cows' milk and to infant formulae containing cow's milk protein are due to the fact that the proteins of cows' milk differ from the proteins of mother's milk and can constitute allergens for humans. The principal recognized cow's milk allergens are alpha-lactalbumin (aLA), beta-lactolglobulin (bLG) and bovine serum albumin (BSA). Bovine whey protein and/or casein are often used as the milk protein source in infant formulae. To reduce allergenicity, cow's milk proteins are hydrolysed by enzymes and thus reduced to peptides. Current hypoallergenic formulas composed of such cow's milk proteins hydrolysates aimed at allergy prevention also comprise other nutrients such as animal oils, vegetable oils, starch, maltodextrin, lactose and sucrose. These protein hydrolysates may also be incorporated into an adult milk drink or food supplements.
The hydrolysis process used to produce these hydrolysates must be carefully monitored so that the final product hydrolysate retains its nutritional value and desired physical properties but is hypoallergenic and tolerogenic.
Hydrolysates may be characterised as “partial” or “extensive” depending on the degree to which the hydrolysis reaction is carried out. Currently there is no agreed legal/clinical definition of Extensively Hydrolyzed Products according to the WAO (World Allergy Organization) guidelines for Cow's milk protein allergy (CMA) but there is agreement that according to the WAO that hydrolyzed formulas have proven to be a useful and widely used protein source for infants suffering from CMA. In the current invention a partial hydrolysate is in one in which 60% of the protein/peptide population has a molecular weight of less than 1000 Daltons, whereas an extensive hydrolysate is one in which at least 95% of the protein/peptide population has a molecular weight of less than 1000 Daltons. These definitions are currently used in the industry. Partial hydrolysates are considered as hypoallergenic (HA) whereas extensive hydrolysates are considered as non allergenic.
Many groups have carried out research in order to optimise the hydrolysis process. Hydrolysis reaction conditions including temperature and reactor volume, number of cycles of hydrolysis, choice of protein substrate, enzyme(s) type and concentration are some of the many factors that influence hydrolysis reaction and thus the physical, chemical and ultimately biological properties of the final product. In EP0353122, mixtures of trypsin and chymotrypsin with a ratio of the trypsin/chymotrypsin activities of 0.33 to 0.66 are used to prepare hypoallergenic whey protein hydrolysates. WO9304593 A1 and U.S. Pat. No. 5,039,532A also disclose a hydrolysis process using trypsin and chymotrypsin, which includes a two-step hydrolysis reaction with a heat denaturation step in between to ensure that the final hydrolysate is substantially free of intact allergenic proteins. The trypsin and chymotrypsin used in these methods are preparations produced by extraction of porcine pancreas. A number of products containing protein hydrolysates prepared based on these methods exist on the market. For example, a Nestlé HA® infant formula may be prepared with a hydrolysate produced using trypsin and chymotrypsin extracted from animal pancreas and its hypoallergenic characteristics have been well studied and documented. Three major papers have been published reporting the results of a large randomized and double-blind intervention study performed in Germany, the aim of which was to compare the effect of hydrolyzed formulas vs standard cow's milk formula for the prevention of allergic manifestations, especially atopic eczema, in at risk infants. The publication are listed as following. The effect of hydrolyzed cow's milk formula for allergy prevention in the first year of life: the German Infant Nutritional Intervention Study, a randomized double-blind trial. von Berg A, Koletzko S, Grübl A, Filipiak-Pittroff B, Wichmann H E, Bauer C P, Reinhardt D, Berdel D; German Infant Nutritional Intervention Study Group. J Allergy Clin Immunol. 2003 March; 111:533-40.
Certain hydrolyzed formulas reduce the incidence of atopic dermatitis but not that of asthma: three-year results of the German Infant Nutritional Intervention Study. von Berg A, Koletzko S, Filipiak-Pittroff B, Laubereau B, Grübl A, Wichmann H E, Bauer C P, Reinhardt D, Berdel D; German Infant Nutritional Intervention Study Group. J Allergy Clin Immunol. 2007 March; 119:718-25.
Preventive effect of hydrolyzed infant formulas persists until age 6 years: long-term results from the German Infant Nutritional Intervention Study (GINI Study). von Berg A, Filipiak-Pittroff B, Kramer U, Link E, Bollrath C, Brockow I, Koletzko S, Grübl A, Heinrich J, Wichmann H E, Bauer C P, Reinhardt D, Berdel D; GINIplus study group, J Allergy Clin Immunodol 2008; 121:1442-7.
The conclusion of the latter GINI study was that early nutritional intervention with Nestlé H.A. infant formula, NAN HA® in high-risk children has a long-term preventive effect on atopic dermatitis until the age of 6 years, indicating a real disease reduction rather than postponement of disease onset.
The long-term preventive effect meaning a preventive effect lasting far beyond the period of feeding Nan HA®, shows that oral tolerance was induced, although the immune mechanisms involved in this tolerance induction have not been examined in the GINI study.
As well as being hypoallergenic it would be highly desirable that the infant formula to be used as a complement to or substitute for breast milk has ability to induce oral tolerance in the infant. Oral tolerance is the specific suppression of cellular and/or humoral immune reactivity to an antigen by prior administration of the antigen by the oral route. It is an important part of the development of the immune system in the first months of life and allows the infant to consume food without adverse reaction. Failure of oral tolerance establishment leads to allergy. The development of oral tolerance is linked to the immune system education, ending up with reduced reaction to food antigens. It is believed that some peptides, that may be specifically present in partially hydrolyzed infant formula, have the ability to interact with the immune system and induce oral tolerance induction. It is believed that these peptides should have particular properties, including a relatively small size, to better support the education of the immune system without acting as allergens themselves. It is also believed that the sequences of the specific peptides may play a significant role. Specific hydrolysate peptidic profiles may actually be at the core of the induction of oral tolerance.
Currently there is a widespread trend to move away from the use of enzymes from animals towards the use of those from microbial sources. There has been much progress in the last 20 years in the area of enzyme production by genetic engineering. This has made it possible to reproducibly prepare large quantities of high quality, high purity enzyme in a relatively short space of time. For these reasons it would be desirable to be able to use enzymes from a microbial source for the production of milk protein hydrolysates to be used in hypoallergenic infant formula. Furthermore it would be highly desirable to that these milk protein hydrolysates have the ability to induce oral tolerance in infants.
Thus in order to retain the hypoallergenic properties of the mammalian enzyme derived hydrolysates those produced with microbial enzymes must possess similar chemical, physical and biological properties. Furthermore, any new infant formula on the market is submitted to strict regulatory guidelines, for example in Europe Directive 2006/141/EC applies. Thus, it is desirable that any new product has a very similar peptide profile to that of the already validated product made with mammalian enzymes in order to keep an allergy preventive effect. Furthermore, it would be highly desirable that these milk protein hydrolysates have the ability to induce oral tolerance in infants.
Similarly, it is desirable to promote the reduction of allergies or adverse effect, enhance the absorption of proteins or amino acid, favour the utilization of proteins or amino acids and/or modulate inflammation processes in sick patients, especially by providing particular hydrolysates of proteins in complete nutritional compositions
There is a need for a hydrolysate of milk-derived proteins prepared by the action of non mammalian enzymes, preferably microbial enzymes that present low allergenicity while presenting the capacity of oral tolerance induction.
There is a need for such and hydrolysate to be preferably comprised in an infant formula and/or a nutritional composition that are targeted to individuals at risk of allergies.
There is a need for such compositions that reduce the risk or severity of allergies later in life and help modulating the occurrence of allergic symptoms.
There is a need for obtaining bacterial enzyme-based hydrolysates with peptidic profiles that share some similarities with hydrolysates obtained from mammalian enzyme and, even more so, that are able to reproduce the oral tolerance properties of the latter, with demonstrated prevention of allergic symptoms.
There is a need to use the same enzymes as above in the preparation of nutritional compositions for sick or fragile patients.
As well as producing “partial” hydrolysates these microbial enzymes can also be used to produce “extensive” protein hydrolysates present in therapeutic formulae, such as those intended to feed cow's milk allergic infants and children. In this case, the target population would be sick (allergic) infants and children that are already sensitized to cow's milk proteins.
Those microbial enzymes could also be used to produce any type of protein hydrolysates used in infant, child, or adult products targeting other benefits than those related to allergy, such as facilitated digestion, enhanced absorption and metabolisation of amino-acids, peptides and proteins, promoted recovery from sickness, optimized utilization of nitrogen sources, promotion of tissue build-up and energy reserve.
To address this problem, the inventors have carried out an extensive research programme, and have compared a number of microbial enzymes as potential candidates for carrying out the hydrolysis reaction. They monitored parameters including hydrolysis reaction performance, enzymes specificity and peptide molecular weight profile and have determined that a number of specific enzyme mixtures provide hydrolysates with the desired physical, chemical and biological properties. The milk protein hydrolysates disclosed herein may be produced efficiently and reproducibly, have an acceptable taste, have the required nutritional value and are hypoallergenic. Furthermore, the hydrolysates of the invention may induce oral tolerance.